PROJECT SUMMARY Trypanosoma brucei, the causative pathogen of trypanosomiasis, threatens >60 million people and causes economic burdens in sub-Saharan Africa. Only a few drugs are available for treating its infection, all with severe side effects and are difficult to administer. Therefore, identification and characterization of essential cellular processes with unique features in T. brucei is essential for developing better anti-parasite agents in the future. DNA replication is essential for cell proliferation and genome integrity. Replication initiation and elongation must coordinate well with nucleosome disassembly and assembly. Importantly, we have discovered that simultaneous deletion of region-specific chromatin marks, histone variants H3v and H4v, and a Kinetoplastid-specific DNA modification, base J, results in severe growth defects. H3v? H4v? J? mutants exhibit replication stress phenotypes, including formation of nuclear TbRPA1 foci (an indicative of abnormal exposure of ssDNA resulting from replication stress), and accumulation of G2 cells and cells with incompletely replicated DNA. Therefore, these chromatin marks are important for proper DNA replication. It is possible that these chromatin marks influence DNA replication only at loci where they normally reside (local effect). It is also possible that changes in chromatin structure may affect DNA replication at regions far away on a linear scale (global effect). In this project, we will test these hypotheses by examining DNA replication at chromosome internal regions (Aim 1) and telomeres (Aim 2) in H3v? H4v? J? mutants. In Aim 1, we will examine origin firing profiles and replication elongation (fork migration pattern) by MFA-seq and Repli-seq in Aim 1.1, examine replication initiation by determining TbORC1 binding at origins in Aim 1.2, determine whether chromatin mark mutations cause chromosome fragility in Aim 1.3 and/or change transcription profile in Aim 1.5. We will also determine the 3D chromosome organization to obtain mechanistic understanding of how chromatin marks control DNA replication inside the nuclear space in Aim 1.4. This will help us to establish logical links between chromatin structure, DNA replication, and transcription status at the chromosome internal regions. T. brucei telomeres provide special benefits for studying chromatin structure, replication, and transcription interplay, because T. brucei subtelomeres are either `highly transcribed and replicated early' or `tightly repressed and replicated late'. These unique features at telomeres will help us unveil additional mechanistic details on the interaction among chromatin structure, replication, and transcription. We will study telomere/subtelomere replication using 2D gel analysis and Chromatin Fiber FISH in Aim 2.1. DNA breaks and recombination will also be examined in Aim 2.2. From this proposal, we will establish foundation to define how chromatin marks work together and control replication and transcription. Our studies will help us understand mechanisms by which this collaborative action between epigenome and genome can successfully channel into genome stability and gene diversification for cellular survival.